PROJECT SUMMARY/ABSTRACT Recent advances in sequencing technology have helped uncover a variety of new driver mutations in malignant melanoma, including recent discoveries of activating mutations in the genes encoding the guanine-nucleotide exchange factor PREX2 and its small GTPase substrate, RAC1. Activating mutations in RAC1 are of special interest, as tumors bearing this mutation are refractory towards current targeted therapeutic agents. The presence of activating PREX2 and RAC1 mutations in a significant fraction of human melanoma suggest that such tumors could be vulnerable to small molecule inhibitors that target its key kinase effectors, such as Group A p21-activated kinases (PAK1, -2, and -3), and phosphatidylinositol-3 kinases, in particular PI3K?. In preliminary studies, we have established that Group A PAKs are critical for oncogenic signaling by RAC1 in a zebrafish model and in RAC1-mutant human melanoma cells. These effects form the basis for Specific Aim 1, in which we use a new method for signaling analysis to establish the activity of the entire kinome in RAC1, BRAF, and NRAS-mutant melanocytes. In Aim 2, we will then test specific PAK and PI3K small molecule inhibitors for their ability to block the effects growth and survival effects of RAC1, as well as determine likely pathways of drug resistance by determining the activity of the kinome before and after drug treatment. In the third aim, we will evaluate a new genetically-engineered mouse model of RAC1-mutant melanoma for use in preclinical studies. Such a model will provide a clinically relevant system to study RAC1 signaling in melanoblast development as well as a rapid testing platform to evaluate therapeutic agents in melanoma.